The balance between oxidants and antioxidants is a key factor for normal brain function (Non Patent Literature 1). The imbalance of redox states caused by an excess of oxidants and/or a depletion of antioxidants is defined as an oxidative-stress state (Non Patent Literature 2). Glutathione is an especially important antioxidant in the central nervous system because of the lower activity of major antioxidant enzymes such as superoxide dismutase and catalase in the brain (Non Patent Literature 3). Glutathione exists in both a reduced form (GSH) and an oxidized form (GSSG), functioning in various redox reactions. Depletion of GSH in the brain is a known cause of neurodegenerative diseases (NDs) such as Parkinson's disease (PD). PD is characterized by a selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) (Non Patent Literature 4). A decrease of GHS is also observed in various diseases such as malignant tumors and infectious diseases and so on.
GSH is a tripeptide composed of cysteine, glutamate and glycine (Non Patent Literature 5). Among these amino acids, cysteine is the rate-limiting factor, since the concentrations of glutamate and glycine in neurons are sufficient. Although cystine is generally known as a source of cysteine, neurons do not express the cystine transport system in mature brains, and thus cysteine is considered a major determinant for intracellular GSH synthesis in neurons.
One of the important factors regulating GSH synthesis is excitatory amino acid carrier 1 (EAAC1), a member of the sodium-dependent excitatory amino acid transporter (EAAT) family. Unlike other EAATs, EAAC1 is selectively enriched in the neurons of the central nervous system (Non Patent Literature 6). It was indicated that the transport of cysteine, rather than that of glutamate, is the major function of EAAC1 (Non Patent Literatures 7 and 8). In fact, EAAC1 deficiency decreased the neuronal GSH content and increased markers of neuronal oxidative stress in the mouse brain (Non Patent Literature 9).
The circadian clock is an internal timekeeping system that allows organisms to adapt physiological and behavioral processes to environmental light/dark cycles (Non Patent Literature 10). Almost all organisms harbor this system, indicating that the circadian clock developed early in the evolution of life. In mammals, the master clock is located in the suprachiasmatic nucleus (SCN). The SCN drive endogenous rhythms and control circadian rhythms in peripheral tissues, including other brain areas such as the SNc (Non Patent Literature 11). The circadian system is regulated by several clock genes such as transcriptional activators (e.g., CLOCK and BMAL1) and repressors (e.g., PER1 and 2). It was shown that BMAL1-deficient mice exhibit increased levels of reactive oxygen species (ROS) and accelerated aging, suggesting that the circadian clock is involved in ROS regulation (Non Patent Literature 12). It was also reported that sleep disorders and circadian disruptions are common in PD patients, and that their symptoms display diurnal fluctuations (Non Patent Literature 13). Together, these reports prompt the interesting theory that there may be a significant correlation between disruption of the circadian system and the misregulation of ROS homeostasis. The mechanism of this association has long been elusive, however.
MicroRNA (miRNA) is a class of small, non-coding molecules that are involved in the post-transcriptional regulation of target gene expression (Non Patent Literature 14). Many miRNAs are highly conserved across species. The sequence in the seed region, which is defined as two to eight nucleotides of miRNA, is the key for determining the target. It has been suggested that miRNAs play important roles in regulating protein levels that exhibit circadian rhythmicity (Non Patent Literature 15). A proteomic analysis in mouse liver revealed that up to 20% of the soluble proteins are rhythmic whereas only 10% of the mRNA is rhythmic (Non Patent Literature 16), suggesting the possible involvement of post-transcriptional regulation such as miRNA regulation. Moreover, several reports have shown that PD-related genes are also regulated by miRNAs (Non Patent Literature 17). Taken together, these findings suggest complicated connections among circadian systems, PD-related gene expression and miRNA regulation, but such connections have not yet been studied.
The present inventors have already proposed a method for screening a substance decreasing an expression of GTRAP3-18 which has a negative function for glutathione synthesis, i.e, a substance promoting glutathione synthesis (Patent Literature 1). As a micro RNA inhibitor and a pharmaceutical composition comprising the inhibitor, for example, a micro RNA inhibitor targeting a gene of tumor-suppression factor and an anti-tumor pharmaceutical composition are known (Patent Literature 2). Regarding to a method for screening a micro RNA inhibitor (antagonist), for example, a screening method of miRNA-29 antagonist is known (Patent Literature 3). In addition, as a nucleic acid medicine such as oligonucleotide, Patent Literature 4 discloses a technique for directing a small interfering RNA (siRNA) to neurons.